Research ArticleCancer

Uncoupling interferon signaling and antigen presentation to overcome immunotherapy resistance due to JAK1 loss in melanoma

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Science Translational Medicine  14 Oct 2020:
Vol. 12, Issue 565, eabb0152
DOI: 10.1126/scitranslmed.abb0152
  • Fig. 1 Jak1 loss, but not Jak2 or Ifnar1 loss, mediates resistance of B16 melanoma to adoptively transferred tumor-specific T cells.

    (A) PD-L1 surface expression by flow cytometry in response to IFN-α, IFN-β, or IFN-γ. (B) Tumor growth (mean ± SEM) of nonirradiated tumors in a dual-flank model. Mice (n = 4 to 6 per group) were treated with focal tumor irradiation (12 Gy) to a contralateral tumor, along with αPD-1 and αCTLA-4 dual ICB (or relevant isotype controls); ***P < 0.001 (unpaired t test). NS, not significant; KO, knockout; Abs, antibodies; XRT, radiation therapy. (C) In vitro and in vivo modeling of efficacy of tumor-specific pmel T cells against B16 wild-type (WT) and CRISPR-modified cell line. (D) In vivo tumor growth (mean ± SEM) of CRISPR-modified B16-F10 tumors (WT, Jak1KO, Jak2KO, and Ifnar1KO) after treatment with ACT. After lymphodepleting total body irradiation (TBI) (5 Gy), tumor-bearing mice (n = 5 per group) were treated with ACT consisting of one dose of 5.0 × 106 pmel (or control BL/6 T cells) along with IL-2 [50,000 IU/day intraperitoneally (i.p.) × 3 days]. **P < 0.01 (repeated-measures two-way ANOVA). (E) In vitro growth (mean ± SD) of CRISPR-modified B16-F10 tumor cell lines pretreated with either IFN-β (left) or IFN-γ (right) and cocultured with tumor-specific pmel T cells (red) or control BL/6 T cells (black). Cocultures performed in biological triplicate; error bars not visible as they are encompassed within the data points. **P < 0.01; ***P < 0.001(unpaired t test).

  • Fig. 2 MHC I expression of human melanoma exhibits IFN-γ dependence.

    (A) Effect of IFN-γ on surface MHC I expression of 48 human melanoma cell lines. Cell lines arranged from left to right by increasing MHC I expression (basal MHC I MFI shown in bottom panel and also as the diameter of the data point in the main panel). Data are shown as log2(fold change) of MFI of the IFN-γ–treated sample relative to untreated control [and also colored from blue to red according to value of log2(fold change)]. (B) Relative change in MFI (mean ± SD) upon IFN-γ exposure as related to basal MHC I expression. *P < 0.05 (unpaired t test). (C) Immunohistochemical staining analysis at baseline and at relapse in a patient with melanoma that developed acquired genetic resistance (loss-of-function JAK1 mutation) to anti–PD-1 ICB. Scale bars, 1 mm (top) and 0.75 mm (bottom). (D) S100 (left) and MHC I (right) expression at the tumor margin in a patient with melanoma that developed acquired genetic resistance to ICB through a loss-of-function mutation in JAK2. Scale bar, 100 μm.

  • Fig. 3 NLRC5 bypasses IFN dependency of B16 MHC I expression and restores sensitivity to adoptive T cell therapy.

    (A) MHC I surface expression by flow cytometry in response to IFN-α, IFN-β, or IFN-γ in CRISPR-modified B16-F10 cell lines. (B) Percentage of MHC I+ cells among CD45RFP+ tumor cells in vivo. Mice (n = 5 to 8 per group) were inoculated subcutaneously with RFP+ tumor cell lines. Established tumors were harvested, and single-cell suspensions were stained for CD45 and MHC I. Box plots depict minimum, maximum, and mean. (C) Surface expression of PD-L1 and MHC I on B16-Jak1KO tumor cells lentivirally transduced with Nlrc5 or empty vector control. Representative samples treated with and without IFN-γ and summary bar plots of MHC I MFI (mean ± SD, n = 3 per group) are shown. ****P < 0.0001. (D) IFN-γ production (mean ± SD) by BL/6 or pmel T cells after 24-hour coculture with indicated tumor cells (which were not pretreated with IFN-γ; n = 3 per group). (E) In vivo tumor growth (mean ± SEM) of B16-Jak1KO-EV and B16-Jak1KO-NLRC5 tumors after treatment with ACT. After lymphodepleting total body irradiation (5 Gy), tumor-bearing mice (n = 5 per group) were treated with ACT consisting of one dose of 5.0 × 106 pmel (or control BL/6 T cells) along with IL-2 (50,000 IU/day i.p. × 3 days). *P < 0.05, ***P < 0.001 (unpaired t test).

  • Fig. 4 BO-112 restores efficacy of tumor-specific T cells against tumor cells lacking both type I and II IFN signaling.

    (A) In vivo growth (mean ± SEM) of B16-Jak1KO tumor cells after treatment with ACT. After lymphodepleting total body irradiation (5 Gy), tumor-bearing mice (n = 4 to 5 mice per group) were treated with ACT consisting of one dose of 5.0 × 106 pmel (or control BL/6 T cells) along with IL-2 (50,000 IU/day i.p. × 3 days). Left: Both groups were treated with intratumoral (i.t.) injection of vehicle control. Right: Both groups were treated with intratumoral injection of BO-112. Intratumoral injections were administered twice a week starting on the day after ACT. *P < 0.05 (unpaired t test). (B) Tumors were harvested 5 days after ACT (and after two doses of intratumoral vehicle or BO-112) for RNA-seq analysis (n = 3 per group). Principal component analysis demonstrates clustering of treatment groups by PC1 and PC3 (treatment groups encircled manually). (C) Top: Venn diagram illustrating the number of genes differentially expressed compared to the control group (BL/6 T cells and vehicle intratumoral agent), highlighting the 700 genes differentially expressed in the group treated with pmel ACT and intratumoral BO-112. The relative expression of this set of 700 genes is shown in the bottom panel colored by treatment group. (D) Heatmap of correlations between the 700 differentially expressed genes from the pmel–BO-112 gene set with CD45+ populations identified by mass cytometry. Red font highlights comparison of adoptively transferred T cells (CD8 pmel T cells) and endogenous T cells (CD8 T cells). (E) In vivo growth (mean ± SEM) of B16-Jak1KO tumors (top, n = 5 to 7 mice per group) or B16-B2mKO tumors (bottom, n = 7 mice per group) after treatment with ACT and intratumoral BO-112. **P < 0.01 (unpaired t test).

  • Fig. 5 BO-112 induces MHC I expression in an IFN- and Nlrc5-independent manner.

    (A) Surface expression of PD-L1 and MHC I (H-2Kb) on B16-WT and B16-Jak1KO cell lines after 18-hour exposure to IFN-β, IFN-γ, or BO-112. (B) Immunofluorescence images of B16-WT and B16-Jak1KO cell lines treated as in (A) and stained with PE-conjugated anti-mouse MHC I (H-2Kb) and 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining, with quantification in the right panel (mean ± SD; n = 48 samples per treatment group). Scale bar, 20 μm. (C) Expression of genes (mean ± SD) involved in MHC I antigen-processing machinery (B2m and Tap1) by quantitative RT-PCR in BO-112–treated B16-Jak1KO tumor cells after 3, 6, and 12 hours, relative to vehicle-treated control (n = 3 per group). (D) IFN-γ production (mean ± SD) by T cells (activated BL6 T cells or pmel T cells) in coculture with B16-WT and B16-Jak1KO tumor cells pretreated with vehicle, BO-112, or IFN-γ (n = 3 per group). (E) Effect of IFN-γ and BO-112 on surface MHC I expression (mean ± SD) of five human melanoma cell lines (n = 2 to 4 per group), including two with known defects in IFN signaling (M202-JAK1KO and M407-JAK1KO) and three cell lines with low basal MHC I expression (see Fig. 2A). (F) B16-Jak1KO tumor cells were modified using CRISPR with guides targeting Nlrc5 to generate two clonal B16-Jak1KO-Nlrc5KO cell lines (cA4.1 and cC5.1). The percentage of MHC I+ in each tumor cell line (mean ± SD) after treatment with IFN-γ or BO-112 for 18 hours (n = 3 per group) is shown. (G and H) MHC I mean fluorescence intensity (mean ± SD) in response to a panel of PRR agonists [LPS (100 ng/ml), CpG (10 μg/ml), poly I:C (100 μg/ml), and BO-112 (0.5 μg/ml)] in mouse (G) and human (H) cell lines (RAW246.7 macrophages, B16-WT and B16-Jak1KO mouse melanoma, and M202-JAK1KO human melanoma; n = 2 to 3 per group). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (unpaired t test).

  • Fig. 6 BO-112 induces IFN and NF-κB gene expression programs in B16-Jak1KO tumor cells despite the absence of IFN signaling.

    (A) B16-Jak1KO tumor cell lines were treated with vehicle or BO-112 for 6 hours, and gene expression was quantified by RNA-seq (n = 3 biological replicates). The left panel shows the top 190 differentially expressed genes [P < 0.01, FDR < 0.05, and log2(fold change) > 1.5], which were also associated with significantly enriched gene sets [P < 0.01, FDR < 0.05, and normalized enrichment score (NES) > 1.5]. The eight enriched gene sets with at least 30 differentially expressed genes are shown in the right panel. (B) Comparison of the 136 genes enriched in tumors treated with BO-112 from our in vivo experiment (left; see also Fig. 4) with genes enriched in B16-Jak1KO tumor cell line treated with BO-112. Relative expression of these 136 genes in the cell lines (right, top) mirrors the expression of these same genes in the groups treated with BO-112 in vivo (right, bottom). (C) Left: Illustration of the in vitro log2(fold change) of 55 genes specific to BO-112 treatment both in vitro and in vivo. Enrichment of type I IFN signaling (blue font) and TNF-α signaling via NF-κB (red font) gene sets is highlighted and shown in larger font. Right: Relative expression of each gene in vitro.

  • Fig. 7 BO-112 bypasses IFN signaling and induces MHC I expression through direct dsRNA sensor-mediated activation of NF-κB signaling.

    (A) Surface expression of MHC I and PD-L1 in B16-Jak1KO tumor cells (representative example) treated with increasing doses of BMS-345541, a selective NF-κB inhibitor. Right: Quantification (mean with individual data points; n = 2 per group). *P < 0.05 (unpaired t test). DMSO, dimethyl sulfoxide. (B) Effect of siRNA targeting Rela on expression of MHC I in mouse B16-Jak1KO (mean, n = 2 per group) and human M407-JAK1KO (mean ± SD; n = 4 per group) tumor cells in response to BO-112. *P < 0.05; **P < 0.01 (unpaired t test). (C) Impact of siRNAs against dsRNA sensors (Pkr, Ifih1, Ddx588, and Tlr3), as well as Rela, on the induction of MHC I by BO-112 (mean ± SD; n = 3 per group). *P < 0.05 (unpaired t test). (D) Effect of siRNA targeting Pkr on expression of nuclear and cytoplasmic NF-κB (p65) in mouse B16-WT and B16-Jak1KO tumor cells in response to BO-112.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/565/eabb0152/DC1

    Fig. S1. Defects in Jak1 or Jak2 do not alter sensitivity of irradiated B16 tumors to irradiation and dual ICB.

    Fig. S2 CRISPR modifications of B16 tumor cell lines do not alter gp100 expression.

    Fig. S3. MHC I expression of human melanoma exhibits IFN-α dependence.

    Fig. S4. MHC I expression of human melanoma exhibits IFN-β dependence.

    Fig. S5. Gating strategy to assess in vivo MHC I expression of B16-F10.

    Fig. S6. Nlrc5 overexpression does not augment the antitumor efficacy of adoptively transferred pmel T cells against B16-WT tumors.

    Fig. S7. BO-112 augments the efficacy of pmel T cells against B16-WT tumors in vivo.

    Fig. S8. BO-112 and pmel ACT alter the immune composition of B16-Jak1KO tumors.

    Fig. S9. B16-B2mKO tumor cells are resistant to killing by pmel T cells.

    Fig. S10. BO-112–induced MHC I up-regulation in B16-Jak1KO cell lines is Nlrc5 independent.

    Fig. S11. BO-112 induces cytoplasmic phosphorylation and nuclear translocation of NF-κB (p65) in B16-Jak1KO cells.

    Fig. S12. Protein-level effects of siRNA targeting Ifih1, Ddx58, or Tlr3.

    Table S1. CRISPR guides and RT-PCR primer sequences.

    Table S2. Reagents.

    Data file S1. Original data.

  • The PDF file includes:

    • Fig. S1. Defects in Jak1 or Jak2 do not alter sensitivity of irradiated B16 tumors to irradiation and dual ICB.
    • Fig. S2 CRISPR modifications of B16 tumor cell lines do not alter gp100 expression.
    • Fig. S3. MHC I expression of human melanoma exhibits IFN-α dependence.
    • Fig. S4. MHC I expression of human melanoma exhibits IFN-β dependence.
    • Fig. S5. Gating strategy to assess in vivo MHC I expression of B16-F10.
    • Fig. S6. Nlrc5 overexpression does not augment the antitumor efficacy of adoptively transferred pmel T cells against B16-WT tumors.
    • Fig. S7. BO-112 augments the efficacy of pmel T cells against B16-WT tumors in vivo.
    • Fig. S8. BO-112 and pmel ACT alter the immune composition of B16-Jak1KO tumors.
    • Fig. S9. B16-B2mKO tumor cells are resistant to killing by pmel T cells.
    • Fig. S10. BO-112–induced MHC I up-regulation in B16-Jak1KO cell lines is Nlrc5 independent.
    • Fig. S11. BO-112 induces cytoplasmic phosphorylation and nuclear translocation of NF-κB (p65) in B16-Jak1KO cells.
    • Fig. S12. Protein-level effects of siRNA targeting Ifih1, Ddx58, or Tlr3.
    • Table S1. CRISPR guides and RT-PCR primer sequences.
    • Table S2. Reagents.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

  • The PDF file includes:

    • Fig. S1. Defects in Jak1 or Jak2 do not alter sensitivity of irradiated B16 tumors to irradiation and dual ICB.
    • Fig. S2 CRISPR modifications of B16 tumor cell lines do not alter gp100 expression.
    • Fig. S3. MHC I expression of human melanoma exhibits IFN-α dependence.
    • Fig. S4. MHC I expression of human melanoma exhibits IFN-β dependence.
    • Fig. S5. Gating strategy to assess in vivo MHC I expression of B16-F10.
    • Fig. S6. Nlrc5 overexpression does not augment the antitumor efficacy of adoptively transferred pmel T cells against B16-WT tumors.
    • Fig. S7. BO-112 augments the efficacy of pmel T cells against B16-WT tumors in vivo.
    • Fig. S8. BO-112 and pmel ACT alter the immune composition of B16-Jak1KO tumors.
    • Fig. S9. B16-B2mKO tumor cells are resistant to killing by pmel T cells.
    • Fig. S10. BO-112–induced MHC I up-regulation in B16-Jak1KO cell lines is Nlrc5 independent.
    • Fig. S11. BO-112 induces cytoplasmic phosphorylation and nuclear translocation of NF-κB (p65) in B16-Jak1KO cells.
    • Fig. S12. Protein-level effects of siRNA targeting Ifih1, Ddx58, or Tlr3.
    • Table S1. CRISPR guides and RT-PCR primer sequences.
    • Table S2. Reagents.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

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